Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 1. JPO2 interacts dynamically with chromatin. A, schematic representation of JPO2 with indication of the known/predicted chromatin and protein- protein interaction domains. B, Western blot of HeLa cells expressing eGFP-JPO2, 24 h post-transfection. The eGFP-JPO2 protein was detected using an eGFP antibody. Non-transfected cells were used as a negative control. C–E, confocal imaging, CP, and FCS analysis of HeLap75KD cells expressing eGFP-JPO2. C, both interphase(top)andmitotic(bottom)cellswereimagedafterstainingforDNAwithDAPI.D,CPcurveswerefittedwithEquation1(solidline).Errorbars,S.D.after normalization. E, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. D and E, CP and FCS data in HeLap75KD cells expressing eGFP or eGFP-LEDGF/p75 are shown for comparison (21). Parameters after fitting are given in Table 1. LZ, leucine zipper; NLS, nuclear localization sequence; au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Imaging, Comparison, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 2. Dynamic chromatin interaction of JPO2 is enhanced by LEDGF/p75. A, schematic representation of LEDGF/p75 with indication of the known chromatin and protein-protein interaction domains. B, Western blot of HeLa cells expressing mRFP-LEDGF/p75, 24 h post-transfection. Endogenous LEDGF/ p75 (L) and mRFP-LEDGF/p75 (mL) were detected using a LEDGF/p75-specific antibody. The minor band below the mRFP-LEDGF/p75 band is due to partial intrachain cleavage of the mRFP-moiety (64). Non-transfected cells were used as a negative control. C and D, confocal imaging and FCS analysis of HeLap75KD cells expressing mRFP-LEDGF/p75. C, both interphase (top) and mitotic (bottom) cells were imaged after staining for DNA with DAPI. D, FCS analysis of eGFP- and mRFP-tagged LEDGF/p75. Average autocorrelation functions and fit with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. Parameters after fitting are depicted in Table 1. The differences between the eGFP- and mRFP-LEDGF/p75 autocorrelation functions in the 10–1000-s time scale are due to the strong blinking contribution in the mRFP1 autocorrelation function (52). E–H, confocal imaging, CP, FCS, and FCCS analysis of HeLap75KD cells co-expressing eGFP-JPO2 and mRFP-LEDGF/p75. E, both interphase (top) and mitotic (bottom) cells were imaged after staining for DNA with DAPI. F, normalizedaverageFCCScross-correlationfunctionfromcellswithequimolarexpressionofeGFP-andmRFP-labeledproteins.Errorbars,S.E.G,CPcurveswere fitted with Equation 1 (solid line). Error bars, S.D. after normalization. H, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. G and H, CP and FCS data in HeLap75KD cells expressing eGFP-LEDGF/p75 (21) and/or eGFP-JPO2 (Fig. 1, D and E) are shown for comparison. Parameters after fitting are depicted in Table 1. au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Imaging, Staining, Comparison

Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 4. LEDGF/p75 trails JPO2 along chromatin via its PWWP domain. A, Western blot of HeLa cells expressing mRFP-LEDGF/p75 (WT) or chromatin binding-deficient mRFP-LEDGF/p75 mutant (K56D/R74D), 24 h post-transfection. Endogenous LEDGF/p75 (L) and mRFP-LEDGF/p75 (mL) were detected using a LEDGF/p75 specific antibody. B–F, confocal imaging, CP, FCS, and FCCS analysis of HeLap75KD cells expressing mRFP-LEDGF/p75K56D/R74D or eGFP-JPO2 mRFP-LEDGF/p75K56D/R74D.BandC,bothinterphase(top)andmitotic(bottom)cellswereimagedafterstainingforDNAwithDAPI.D,CPcurveswerefittedwith Equation 1 (solid line). Error bars, S.D. after normalization. E, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. D and E, CP and FCS data in HeLap75KD cells expressing eGFP-JPO2 (Fig. 1D) or eGFP-JPO2 mRFP-LEDGF/p75 (Fig. 2, G and H) are shown for comparison. Parameters after fitting are given in Table 1. F, box plot of the CCrel values from different cells (number of cells 6) with equimolar expression of eGFP- and mRFP-labeled proteins. Whiskers represent the 5th and 95th percentile. *, statistically significant difference (p 0.01 from an unpaired Student’s t test). FCCS data from Fig. 2F are shown for comparison. au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Binding Assay, Mutagenesis, Transfection, Imaging, Comparison, Labeling

Journal: Cell Reports

Article Title: Systemic IL-15, IFN-γ, and IP-10/CXCL10 signature associated with effective immune response to SARS-CoV-2 in BNT162b2 mRNA vaccine recipients

doi: 10.1016/j.celrep.2021.109504

Figure Lengend Snippet:

Article Snippet: SpectraMax M3 , Molecular Devices, LLC , N/A.

Techniques: Recombinant, Biomarker Assay, Neutralization, Luciferase, Cell Culture, Lysis, Expressing, Software

Journal: Pharmacological research

Article Title: Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

doi: 10.1016/j.phrs.2018.09.005

Figure Lengend Snippet: Fig. 4. Effect of CORM-401 and PAPA NONOate on MDA-MB-231-luc2-tdTomato cell transendothelial migration and adhesion to lung microvascular endothelial cells.

Article Snippet: Human lung microvascular endothelial cell line (hLMVEC) was purchased from the European Cell Culture Collection (Cell Applications, San Diego, CA, USA), human breast adenocarcinoma MDA-MB-231-luc2-tdTomato cell line stably expressing the firefly luciferase gene and tdTomato fluorescent protein was kindly provided by Prof. Joanna Wietrzyk (Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences). hLMVEC cells were maintained in Microvascular Endothelial Cell Growth Medium (Cell Applications), MDA-MB-231-luc2-tdTomato cells were cultured in RPMI1640 GlutaMAX Medium (Gibco) with 10% FBS (Gibco), AAS (Sigma-Aldrich) containing 20 units of penicillin, 20 mg streptomycin and 0.05 mg amphotericin B.

Techniques: Migration

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control